Glycine is the major inhibitory neurotransmitter in brainstem and spinal cord, whereas in hippocampus glycine exerts dual modulatory roles on strychnine-sensitive glycine receptors and on the strychnine-insensitive glycineB site of the N-methyl-D-aspartate receptor (NMDAR). In hippocampus, the synaptic availability of glycine is largely under control of glycine transporter 1 (GlyT1). Since epilepsy is a disorder of disrupted network homeostasis affecting the equilibrium of various neurotransmitters and neuromodulators, we hypothesized that changes in hippocampal GlyT1 expression and resulting disruption of glycine homeostasis might be implicated in the pathophysiology of epilepsy. Using two different rodent models of temporal lobe epilepsy (TLE) – the intrahippocampal kainic acid model of TLE in mice, and the rat model of tetanic stimulation-induced TLE – we first demonstrated robust overexpression of GlyT1 in the hippocampal formation, suggesting dysfunctional glycine signaling in epilepsy. Overexpression of GlyT1 in the hippocampal formation was corroborated in human TLE samples by quantitative real time PCR. In support of a role of dysfunctional glycine signaling in the pathophysiology of epilepsy, both the genetic deletion of GlyT1 in hippocampus and the GlyT1 inhibitor LY2365109 increased seizure thresholds in mice. Importantly, chronic seizures in the mouse model of TLE were robustly suppressed by systemic administration of the GlyT1 inhibitor LY2365109. We conclude that GlyT1 overexpression in the epileptic brain constitutes a new target for therapeutic intervention, and that GlyT1 inhibitors constitute a new class of antiictogenic drugs. These findings are of translational value since GlyT1 inhibitors are already in clinical development to treat cognitive symptoms in schizophrenia.
Hematopoietic progenitor kinase 1 (HPK1, MAP4K1) is a serine/threonine kinase that has been demonstrated to have suppressive effects across a range of immune cells, including T cells and dendritic cells. Loss of MAP4K1 kinase activity is sufficient to enhance T cell receptor (TCR) signaling resulting in robust anti-tumor immunity alone and in combination with checkpoint inhibition. These data support that MAP4K1 is a novel and attractive target for cancer immunotherapy. We have designed a series of potent, selective, and orally bioavailable inhibitors of MAP4K1. Treatment of primary human T cells or peripheral blood with either BLU2069 or BLU6348 was able to inhibit phosphorylation of pSLP76, a scaffolding protein that regulates MAPK downstream of the TCR. In addition, we show that compound treatment can enhance cytokine secretion and proliferation in human T cells in response to TCR crosslinking. The therapeutic benefit of MAP4K1 inhibition alone and in combination with anti-PD-L1 was evaluated in multiple syngeneic mouse tumor models including MCA205, MC38 and EMT-6. Treatment with either compound alone led to a reduction in tumor growth that was further enhanced when combined with anti-PD-L1 therapy. When tumors were grown in immunocompromised mice (MCA-205) or in the setting of CD8+ T cell depletion (MC-38), the anti-tumor effect of BLU2069 and BLU6348 respectively was lost, confirming the importance of immune cells in compound mediated antitumor effects. We further show that MCA205 tumors harvested from mice treated with BLU2069 had increased intratumoral CD8+ T cell infiltration, resulting in enhanced CD8/Treg ratios. In addition, transcriptional analysis of tumor lysates showed that BLU2069 significantly increased genes associated with an effector phenotype. These data support that pharmacological inhibition of MAP4K1 reduced tumor burden and enhanced antitumor immunity in preclinical tumor models. Finally, we show that MAP4K1 inhibition can enhance CD3/CD28-induced IL2 and IFNγ in human tumor infiltrating lymphocytes (TILs) generated from melanoma or non-small cell lung cancer (NSCLC) primary tumors. This work describes the identification of potent small molecule inhibitors of MAP4K1 which could be novel therapeutic agents and induce an effective immune response either alone or in combination with approved checkpoint inhibitors. Citation Format: Kerrie Faia, Alberto Toso, Kristina Fetalvero, Marly Roche, Steven Bench, Erin O'Hearn, Qiongfang Cao, Kerry-Ann Bright, Debora Paduraru, Andrea Romagnani, Weifan Weng, Tina Zimmermann, Michael Burke, Joshua Close, Luke Green, Joseph Kim, Chandra Miduturu, Alison Ribeiro, Marina Bacac, Sylvia Herter, Emanuele Perola, Michael Sheets, Jan Eckmann, Gordon Heidkamp, Tary Traore, Erik Gerson, Rich Woessner, Carsten Wolter, Felix Scheuplein, Nisha Perez, Timothy LaBranche, Grace Silva, Chaoyang Ye, Caitlin Utt, Stefan Gross, James R. Bischoff, Marion Dorsch, Tim Guzi, Klaus Hoeflich, Jason Brubaker. MAP4K1 inhibition enhances immune cell activation and anti-tumor immunity in preclinical tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1717.
Genomic assault in the form of DNA double-strand breaks represents the most efficient mechanism by which radiotherapy mediates killing of cancer cells. In order to preserve cellular integrity and mitigate the adverse effects and complications arising from insult to healthy tissues, a fractionated dosing regimen, where low doses of ionizing radiation are administered over a course of several days, is the principal mode of radiation therapy in clinical settings. Additionally, it has been demonstrated that fractionated dosing has a greater capacity to elicit anti-tumor activity and immune mediated abscopal response, compared to bolus delivery. However, in most preclinical animal models of cancer that employ the use of focal beam radiation, administration of a single bolus dose is the standard practice. To establish a more translationally relevant approach to radiation therapy, we utilized computerized tomography (CT) guided Small Animal Radiation Research Platform (SARRP) in three murine subcutaneous tumor models, MB-49 (bladder), B16F10 (melanoma) and MC38 (colon). Each treatment cohort was subjected to a fractionated dosing regimen, and response was assessed by caliper measurements of tumor volume. Therapy was well tolerated across all models and dosing regimens, as evidenced by no significant body weight loss or presentation of adverse clinical symptoms compared to controls. Fractionated focal radiation at 2Gy delivered over a course of five consecutive days (QDx5) induced a modest anti-tumor response in the MB-49 model, resulting in a 66.7% increase in time to progression (ITP) and a 64.1% median ΔT/ΔC on day 16 post implant. Mice bearing MC38 tumors exhibited meaningful response with incidences of 55% ITIP and a median ΔT/ΔC of 29% on day 26, following treatment with radiation at 2Gy, QDx5. Additionally, fractionated doses at 10Gy and 5Gy both delivered Q5Dx2 produced robust anti-tumor response in the B16F10 model, with incidences of 5% and 21% median ΔT/ΔC on Day 15, respectively. Though notable responses were observed across all treatment cohorts, there were no incidences of tumor free survivors or complete regressions, thus allowing for additional therapeutic intervention and synergistic approaches. Further assessment of fractionated dosing regimens in additional mouse models, as well as potential impact on immunomodulation, are ongoing. Over 50% of cancer patients are treated with radiotherapy, with a significant portion requiring integration with other therapeutic modalities to enhance survival and curative benefits. It is therefore imperative to identify appropriate fractionated dose levels and schedules across preclinical cancer models, in order to more accurately reflect the clinical landscape and provide a more viable framework for interrogating rational combination strategies. Citation Format: Kerry-Ann Bright, Derrik Germain, Erin Trachet, Sheri Barnes. Fractionated dosing: A more clinically relevant approach to radiotherapy in preclinical tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2402.
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